Monoterpene cyclases (synthases) provide the focus for study of allylic pyrophosphate cyclization, a reaction type of major importance in C-C bond formation in the biosynthesis of numerous terpenoid natural products of pharmacological significance. A general stereochemical model has been proposed for the coupled isomerization-cyclization of the universal isoprenoid precursor, geranyl pyrophosphate, and key mechanistic elements of the scheme were confirmed through studies on the origin of the seven major monoterpene skeletal types. A selected set of cyclases [(+)- and (- )limonene synthase, (+)- and (-)-pinene synthase, and (+)- and (-)-bornyl pyrophosphate synthase] that differ significantly in mechanistic detail will be employed to examine active site structure-function relationships that underlie reaction variants. As the prototype, (-)-4S-limonene synthase was purified from isolated oil glands of Mentha and, from amino acid sequence information, degenerate oligonucleotide probes were prepared for screening an oil gland cDNA library, from which three full-length clones were isolated. These cDNA isolates were sequenced and verified by functional expression in Escherichia coil. Based on positive RNA blot hybridization and direct sequence comparison at the protein level, the (- )-limonene cyclase cDNA provides a powerful heterologous probe for isolating the cDNAs encoding the other monoterpene cyclases from the corresponding gland libraries of Salvia, Tanacetum and Citrus species. Where heterologous cDNA probing is not possible, an alternate strategy for purifying the target cyclase, and obtaining the gene, has been devised based on specific labeling of the protein with a mechanism-based inhibitor. A bacterial overexpression system based on the PET vector will be devised that permits rapid purification of the recombinant cyclases. The roles of active site cysteine and histidine residues in binding and catalysis were suggested by inhibition studies and by determining the protective influence of substrate and analogues representing different substrate binding domains. A photolabile substrate analogue was utilized for photoaffinity labeling of the presumptive hydrophobic pocket of the cyclases, and the mechanism-based inhibitor was used for labeling putative active site bases involved in terminating deprotonations. With these probes, labeled active site peptides will be generated for sequencing and location on the deduced primary structures. This information, plus that gained by primary sequence comparisons, will be used to target active site residues for mutagenesis. The mutant cyclases will be characterized with respect to kinetic behavior and product mixture, and a variety of biochemical techniques employed to deduce which step(s) of the complex reaction cascade have been altered. A series of substrate analogues will be used to examine the cryptic isomerization step of the reaction and to explore the catalytic repertoire of the cloned cyclases. The studies outlined should provide new information on the nature of these novel catalysts, particularly the relationship of enzyme structure to reaction mechanism, and allow a clearer understanding of this important aspect of prenyl pyrophosphate metabolism.